The long-term goal of this research is to elucidate the molecular and cellular mechanisms that ensure potassium (K+) channels assemble with the appropriate membrane-embedded regulatory subunits for proper physiological function in the heart, muscle, and nerve. In the previous funding period, we developed an innovative approach to label the t-tubules and sarcolemma of cardiomyocytes with either small molecule or protein-based K+-sensitive fluorophores. For this proposal, we will use our fluorescent bioconjugates to test the long standing hypothesis that K+ accumulates faster in the t-tubules to protect the heart during physical activity (Aim 1); to fluorescently visualize specific ion channel activity in order to identify the K+ channel/KCNE complexes responsible for maintaining cardiac rhythmicity (Aim 2). The completion of these aims will yield a transformative set of methodologies to directly investigate extracellular K+ accumulation from heart, muscle, and nerve cells isolated from healthy and disease animal models.